Popova Elena, Bader Michael, Krivokharchenko Alexander
Max-Delbrück Center for Molecular Medicine (MDC), D-13092 Berlin-Buch, Germany.
Biol Reprod. 2006 Oct;75(4):524-30. doi: 10.1095/biolreprod.106.053330. Epub 2006 Jun 28.
Cloning technology would allow targeted genetic alterations in the rat, a species which is yet unaccessible for such studies due to the lack of germline-competent embryonic stem cells. The present study was performed to examine the developmental ability of reconstructed rat embryos after transfer of nuclei from early preimplantation stages. We observed that single blastomeres from two-cell embryos and zygotes reconstructed by pronuclei exchange can develop in vitro until morula/blastocyst stage. When karyoplasts from blastomeres were used for the reconstruction of embryos, highest in vitro cleavage rates were obtained with nuclei in an early phase of the cell cycle transferred into enucleated preactivated oocytes or zygotes. However, further in vitro development of reconstructed embryos produced from blastomere nuclei was arrested at early cleavage stages under all conditions tested in this study. In contrast, immediate transfer to foster mothers of reconstructed embryos with nuclei from two-cell embryos at an early stage of the cell cycle in preactivated enucleated oocytes resulted in live newborn rats, with a general efficiency of 0.4%-2.2%. The genetic origin of the cloned offspring was verified by using donor nuclei from embryos of Black Hooded Wistar rats and transgenic rats carrying an ubiquitously expressed green fluorescent protein transgene. Thus, we report for the first time the production of live cloned rats using nuclei from two-cell embryos.
克隆技术将使对大鼠进行靶向基因改造成为可能，由于缺乏具有种系能力的胚胎干细胞，目前该物种还无法进行此类研究。本研究旨在检测植入前早期阶段的细胞核移植后重构大鼠胚胎的发育能力。我们观察到，通过原核交换重构的二细胞胚胎和受精卵的单个卵裂球能够在体外发育至桑葚胚/囊胚阶段。当使用卵裂球的核质体进行胚胎重构时，将处于细胞周期早期的细胞核转移至去核的预激活卵母细胞或受精卵中，体外分裂率最高。然而，在本研究测试的所有条件下，由卵裂球细胞核产生的重构胚胎的进一步体外发育在早期分裂阶段就停止了。相比之下，将处于细胞周期早期的二细胞胚胎细胞核移植到预激活的去核卵母细胞中，重构胚胎立即移植到代孕母鼠体内，产下了活的新生大鼠，总体效率为0.4%-2.2%。通过使用来自黑帽Wistar大鼠胚胎和携带普遍表达的绿色荧光蛋白转基因的转基因大鼠的供体细胞核，验证了克隆后代的遗传来源。因此，我们首次报道了使用二细胞胚胎细胞核生产活的克隆大鼠。
